The present invention relates to the leukocyte adhesion receptor Mac-1. The invention further pertains to the cloning of DNA sequences which encode the alpha-subunit of this molecule. This invention was made in part with government support. The government has certain rights in this invention.
The immune system is responsible for protecting an animal from foreign invaders, such as bacteria, viruses, etc. An excellent review of the defense system is provided by Eisen, H. W. (In: Microbiology, 3rd Ed., Harper and Row, Philadelphia, Pa. (1980), pp. 290-295 and 381-418). The ability of the immune system to protect an animal against foreign invaders depends, in large measure, on the presence and function of blood cells known an leukocytes. The ability of leukocytes to provide such protection has been found to require that these cells adhere to cellular and extracellular substrates.
For example, leukocytes must be able to attach to endothelial cells so that they can migrate from circulation to sites of ongoing inflammation. Furthermore, they must attach to antigen-presenting cells so that a normal immune response can occur. They must also be able to attach to appropriate target cells so that the lysis of virally-infected (or tumor) cells can occur. Furthermore, leukocytes must be able to attach to various activated proteins (such as iC3b-the activated form of the third component of complement) so that they may efficiently phagocytose and clear microbial and cellular debris. Thus, leukocyte adhesion is a requisite of a normally functioning host defense system. The inhibition of this defense system is desirable in cases such as transplantation, because the host xe2x80x9cseesxe2x80x9d the transplanted tissue as foreign and initiates an immune response to that tissue. Leukocyte adhesion is, therefore, also involved in the rejection of transplanted tissue and organs. Thus, an understanding of leukocyte adhesion may enable one to either augment an animal""s ability to fight infection or suppress an animal""s capacity to reject transplanted tissue.
Recently, leukocyte surface molecules involved in mediating leukocyte adhesion were identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T-cells (Davignon, D., et al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleen cells (Springer, T., et al., Eur. J. Immunol. 9:301-306 (1979)) were identified which bound to leukocyte surfaces and inhibited the attachment-related functions described above (Springer, T., et al., Fed. Proc. 44:2660-2663 (1985)). The molecules which were recognized by these antibodies comprise a set of leukocyte adhesion receptors known as the xe2x80x9cLymphocyte Function-Associated Antigen-1 familyxe2x80x9d (or the xe2x80x9cLFA-1 familyxe2x80x9d) of adhesion receptor molecules.
The LFA-1 family of adhesion receptor molecules contains three highly related cell surface glycoproteins. These glycoproteins have been found to mediate cell-cell interactions in inflammation. The glycoproteins have been designated xe2x80x9cLFA-1xe2x80x9d (lymphocyte function-associated antigen-1), xe2x80x9cMac-1xe2x80x9d and xe2x80x9cp150,95.xe2x80x9d Whereas LFA-1 is found on the surfaces of most leukocytes (Springer, T. A., et al., Immunol. Rev. 68:111-135 (1982)), Mac-1 and p150,95 are found primarily on macrophages, granulocytes and other large granular lymphocytes (Springer, T. A., et al., Immunol. Rev. 68:111-135 (1982); Keizer, G., et al., Eur. J. Immunol. 15:1142-1147 (1985)).
The LFA-1 glycoprotein family is composed of heterodimers, each containing an alpha-subunit which is non-covalently associated with a beta-subunit. The alpha-subunits of the family have been found to differ from one another and are designated CD11a, CD11b, and CD11c, respectively. The glycosylated alpha-subunits have approximate molecular weights of 180, 170, and 150 kd, respectively. In contrast, the beta-subunit of the LFA-1 family of adhesion receptors has been found to be identical, and to have a molecular weight of 95 kd (Sanchez-Madrid, F., et al., J. Exper. Med. 158:1785-1803 (1983); Keizer, G. D., et al., Eur. J. Immunol. 15:1142-1147 (1985); Springer, T., Fed. Proc. 44:2660-2663 (1985); Sanchez-Madrid, F., et al., J. Exper. Med. 158:586-602 (1983)).
Although the alpha-subunits of the glycoproteins do not exhibit the extensive homology shared by the beta-subunits, close analysis of the alpha-subunits of the glycoproteins has revealed that there are substantial similarities between them. Reviews of the similarities between the alpha and beta-subunits of the adhesion molecule glycoprotein family are provided by Sanchez-Madrid, F., et al. (J. Exper. Med. 158:586-602 (1983); J. Exper. Med. 158:1785-1803 (1983); Miller, L. J., et al., J. Immunol. 138:2381-2383 (1987)).
The importance of the LFA-1 family of receptors was initially recognized in studies which showed the ability of monoclonal anti-bodies (which were capable of binding to either the specific alpha-subunits, or the common beta-subunit) to inhibit adhesion-dependent leukocyte functions (Sanchez-Madrid, F., et al., Proc. Natl. Acad. Sci. USA 79:7489-7493 (1982); Beller, D. I., et al., J. Exper. Med. 156:1000-1009 (1982)).
Recently, a group of individuals has been identified who are unable to express normal amounts of any member of the Mac-1 adhesion protein family on their leukocyte cell surfaces. This disease has been termed xe2x80x9cLeukocyte Adhesion Deficiencyxe2x80x9d or xe2x80x9cLADxe2x80x9d and is characterized by chronic and recurring infections, as well as other clinical symptoms (Anderson, D. C., et al., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C., et al., J. Infect. Dis. 152:668-689 (1985)). Leukocytes from LAD patients display in vitro defects which were similar to those observed when leukocytes of normal individuals were antagonized by antibody specific for members of the LFA-1 family. LAD patients were found to be unable to mount a normal immune response. This failure was found to be due to an inability of the leukocytes of LAD patients to adhere to cellular and extracellular substrates (Anderson, D. C., et al., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C., et al., J. Infect. Dis. 152:668-689 (1985)). These studies show that inflammatory reactions are mitigated when leukocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules on their cell surface.
Thus, in summary, the ability of leukocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells) and proteins (such as iC3b). This adherence has been found to require contacts which involve specific receptor molecules present on the leukocyte surface of the leukocytes. These cell surface receptor molecules have been found to be highly related to one another. Humans whose leukocytes lack these cell surface receptor molecules exhibit chronic and recurring infections, as well as other clinical symptoms.
Since leukocyte adhesion is involved in the process through which foreign tissue is identified and rejected, an understanding of this process is of significant value in the fields of organ transplantation, tissue grafts, allergy and oncology.
The present invention relates to leukocyte cell surface adhesion receptor molecules, and in particular, to the cloning and expression of the alpha-subunit of the Mac-1 receptor molecule through the use of recombinant DNA technology. The invention pertains to the adhesion molecule itself, to functional fragments of the molecule, to nucleic acid (i.e., DNA, and especially cDNA) capable of encoding these receptor molecules, and to plasmids which contain such nucleic acid sequences. The present invention additionally encompasses methods for producing the receptor molecules which employ recombinant DNA technology.
In detail, the invention pertains to Mac-1 alpha-subunit, or a functional derivative thereof, substantially free of natural contaminants.
The invention further pertains to the above Mac-1 alpha-subunit or the functional derivative thereof, which is additionally capable of binding to a molecule present on the surface of a cell.
The invention also includes the above Mac-1 alpha-subunit molecule, wherein the molecule contains at least one polypeptide selected from the group consisting of:
The invention also includes a recombinant DNA molecule capable of expressing either the Mac-1 alpha-subunit or a functional derivative thereof.
The invention also provides a method for recovering Mac-1 alpha-subunit in substantially pure form which comprises the steps:
(a) solubilizing Mac-1 alpha-subunit from the membranes of cells expressing Mac-1 alpha-subunit, to form a solubilized Mac-1 alpha-subunit preparation,
(b) introducing the solubilized Mac-1 alpha-subunit preparation to an affinity matrix, the matrix containing immobilized antibody capable of binding to Mac-1 alpha-subunit,
(c) permitting the Mac-1 alpha-subunit to bind to the antibody of the affinity matrix,
(d) removing from the matrix any compound incapable of binding to the antibody and
(e) recovering the Mac-1 alpha-subunit in substantially pure form by eluting the Mac-1 alpha-subunit from the matrix.
The invention also provides a method for treating inflammation resulting from a response of the non-specific defense system (such as asthma; adult respiratory distress syndrome; multiple organ injury syndrome secondary to septicemia; multiple organ injury syndrome secondary to trauma; reperfusion injury of tissue; acute glomerulonephritis; reactive arthritis; dermatosis with acute inflammatory components; a central nervous system inflammatory disorder; thermal injury; hemodialysis; leukapheresis; ulcerative colitis; Crohn""s disease; necrotizing enterocolitis; granulocyte transfusion associated syndrome; and cytokine-induced toxicity) in a mammalian subject which comprises providing to a subject in need of such treatment an anti-inflammatory agent, in an amount sufficient to suppress said inflammation; wherein said anti-inflammatory agent is selected from the group consisting of: the Mac-1 alpha-subunit; and a functional derivative of the Mac-1 alpha-subunit.
The invention also pertains to the above method which additionally comprises the co-administration of an agent selected from the group consisting of: the Mac-1 beta-subunit, and a functional derivative of the Mac-1 beta-subunit.